The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Certain cells in the body respond not only to chemical signals, but also to ions such as extracellular calcium ions (Ca.sup.2+). Changes in the concentration of extracellular Ca.sup.2+ (referred to herein as "[Ca.sup.2+ ]") alter the functional responses of these cells. One such specialized cell is the parathyroid cell -which secretes parathyroid hormone (PTH). PTH is the principal endocrine factor regulating Ca.sup.2+ homeostasis in the blood and extracellular fluids.
PTH, by acting on bone and kidney cells, increases the level of Ca.sup.2+ in the blood. This increase in [Ca.sup.2+ ] then acts as a negative feedback signal, depressing PTH secretion. The reciprocal' relationship between [Ca.sup.2+ ] and PTH secretion forms the essential mechanism maintaining bodily Ca.sup.2+ homeostasis.
Extracellular Ca.sup.2+ acts directly on parathyroid cells to regulate PTH secretion. The existence of a parathyroid cell surface protein which detects changes in [Ca.sup.2+ ] has been suggested. This protein acts as a receptor for extracellular Ca.sup.2+ ("the calcium receptor"), and is suggested to detect changes in [Ca.sup.2+ ] and to initiate a functional cellular response, PTH secretion. For example, the role of calcium receptors and extracellular Ca.sup.2+ in the regulation of intracellular Ca.sup.2+ and cell function is reviewed in Nemeth et al., Cell Calcium 11: 319, 1990; the role of calcium receptors in parafollicular and parathyroid cells is discussed in Nemeth, Cell Calcium 11: 323, 1990; and the role of calcium receptors on bone osteoclasts is discussed by Zaidi, Bioscience Reports 10: 493, 1990.
Other cells in the body, specifically the osteoclast in bone, the juxtaglomerular, proximal tubule cells in the kidney, the keratinocyte in the epidermis, the parafollicular cell in the thyroid, intestinal cells, and the trophoblast in the placenta, have the capacity to sense changes in [Ca.sup.2+ ]. It has been suggested that cell surface calcium receptors may also be present on these cells, imparting to them the ability to detect and to initiate or enable a response to changes in [Ca.sup.2+].
In-parathyroid cells, osteoclasts, parafollicular cells (C-cells); keratinocytes, juxtaglomerular cells, trophoblasts, pancreatic beta cells and fat/adipose cells, an increase in [Ca.sup.2+ ] evokes an increase in intracellular free Ca.sup.2+ concentration ("[Ca.sup.2+ ].sub.i "). Such an increase may be caused by influx of extracellular Ca.sup.2+ or by mobilization of Ca.sup.2+ from intracellular organelles. Changes in [Ca.sup.2+ ].sub.i are readily monitored and quantitated using fluorimetric indicators such as fura-2 or indo-1 (Molecular Probes, Eugene, Oreg.). Measurement of [Ca.sup.2+ ].sub.i provides an assay to assess the ability of molecules to act as agonists or antagonists at the calcium receptor.
In parathyroid cells, increases in the concentration of extracellular Ca.sup.2+ evoke rapid and transient increases in [Ca.sup.2+ ], which are followed by lower, yet sustained, increases in [Ca.sup.2+ ].sub.i. The transient increases in [Ca.sup.2+ ].sub.i arise from the mobilization of intracellular Ca.sup.2+, whereas the lower, sustained increases result from the influx of extracellular Ca.sup.2+. The mobilization of intracellular Ca.sup.2+ is accompanied by increased formation of inositol-1,4,5-triphosphate (IP.sub.3) and diacylglycerol, two biochemical indicators which are associated with receptor-dependent mobilization of intracellular Ca.sup.2+ in various other cells.
In addition to Ca.sup.2+, various other di- and trivalent cations, such as Mg.sup.+, Sr.sup.2+, Ba.sup.2+, La.sup.3+ and Gd.sup.3+ also cause the mobilization of intracellular Ca.sup.2+ in parathyroid cells. Mg+and La.sup.3+ also increase the formation of IP.sub.3. All of these inorganic cations depress the secretion of PTH. The postulated calcium receptor on the parathyroid cell is therefore promiscuous because it detects a variety of extracellular di- and trivalent cations.
The ability of various compounds to mimic extracellular Ca.sup.2+ in vitro is discussed by Nemeth et al., (spermine and spermidine) in "Calcium-Binding Proteins in Health and Disease," 1987, Academic Press, pp. 33-35; Brown et al., (e.g., neomycin) Endocrinology 128: 3047, 1991; Chen et al., (diltiazem and its analog, TA-3090) J. Bone and Mineral Res. 5: 581, 1990; and Zaidi et al., (verapamil) Biochem. Biophys. Res. Commun. 167: 807, 1990.
Brown et al., J. Bone Mineral Res. 6: 11, 1991 discuss theories regarding the effects of Ca.sup.2+ ions on parathyroid cells, and propose that the results may be explained by both a receptor-like mechanism and a receptor-independent mechanism as follows:
Polyvalent cations [e.g., divalent and trivalent cations] exert a variety of effects on parathyroid function, such as inhibition of parathyroid hormone (PTH) secretion and cAMP accumulation, stimulation of the accumulation of inositol phosphates, and elevation of the cytosolic calcium concentration. These actions are thought to be mediated through a "receptor-like" mechanism. The inhibition of agonist-stimulated cAMP accumulation by divalent and trivalent cations, for example, is blocked following preincubation with pertussis toxin. Thus, the putative polyvalent cation receptor may be coupled to inhibition of adenylate cyclase by the inhibitory guanine nucleotide regulatory (G) protein, G.sub.i. PA1 where each X independently is selected from the group consisting of H, CH.sub.3, CH.sub.3 O, CH.sub.3 CH.sub.2 O, methylene dioxy, Br, Cl, F, I, CF.sub.3, CHF.sub.2, CH.sub.2 F, CF.sub.3 O, CF.sub.3 CH.sub.2 O, CH.sub.3 S, OH, CH.sub.2 OH, CONH.sub.2, CN, NO.sub.2, CH.sub.3 CH.sub.2, propyl, isopropyl, butyl, isobutyl, t-butyl, and acetoxy; PA1 Ar is a hydrophobic entity; PA1 each R independently is selected from the group consisting of hydrogen, methyl, ethyl, propyl, isopropyl, allyl, butyl, isobutyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, indenyl, indanyl, dihydroindolyl, thiodihydroindolyl, and 2-, 3-, or 4- piperid(in)yl; PA1 Y is selected from the group consisting of CH, nitrogen and an unsaturated carbon; and PA1 Z is selected from the group consisting of oxygen, nitrogen, sulfur, ##STR2## PA1 where each n is independently between 1 and 4 inclusive; and PA1 each m is independently between 0 and 5 inclusive. PA1 where alk is straight- or branched-chain alkylene of from 0 to 6 carbon atoms; PA1 R.sub.1 is lower alkyl of from 1 to 3 carbon atoms or lower haloalkyl of from 1 to 3 carbon atoms substituted with from 1 to 7 halogen atoms; PA1 R.sub.2 and R.sub.3 are independently selected carbocyclic aryl or cycloalkyl groups, either monocyclic or bicyclic, having 5- to 7-membered rings optionally substituted with 1 to 5 substituents independently selected from lower alkyl of 1 to 3 carbon atoms, lower haloalkyl of 1 to 3 carbon atoms substituted with 1 to 7 halogen atoms, lower alkoxy of 1 to 3 carbon atoms, halogen, nitro, amino, alkylamino, amido, lower alkylamido of 1 to 3 carbon atoms, cyano, hydroxy, acyl of 2 to 4 carbon atoms, lower hydroxyalkyl of 1 to 3 carbon atoms or lower thioalkyl of 1 to 3 carbon atoms. Suitable carbocyclic aryl groups are groups having one or two rings, at least one of which has aromatic character and include carbocyclic aryl groups such as phenyl and bicyclic carbocyclic aryl groups such as naphthyl. PA1 where each X is preferably independently selected from the group consisting of Cl, F, I, CF.sub.3, CH.sub.3, isopropyl, CH.sub.3 O, CH.sub.3 S, CF.sub.3 O, CF.sub.3 CH.sub.2 O, an aliphatic ring and an attached or fused, preferably fused aromatic ring. Preferably, the aromatic and aliphatic rings have 5 to 7 members. More preferably, the aromatic and aliphatic rings contain only carbon atoms (i.e., the ring is not a heterocyclic ring); and PA1 R is preferably H, CH.sub.3, ethyl, or isopropyl.
We recently showed that the polycationic antibiotic, neomycin, mimics the actions of di- and trivalent cations in several aspects of parathyroid function. To determine whether these actions were specific to this agent or represented a more generalized action of polycations, we tested the effects of the highly basic peptides, polyarginine and polylysine, as well as protamine on the same parameters in dispersed bovine parathyroid cells. The results demonstrate that the parathyroid cell responds to a variety of polycations as well as to polyvalent cations, potentially via similar biochemical pathways. These results are discussed in terms of the recently postulated, "receptor-independent" modulation of G proteins by polycations in other systems.
The Ca.sup.2+ receptor has,been presumed to be analogous to other G protein-coupled receptors [e.g., a glycoprotein], but recent studies with other cell types have raised the possibility that polycations can modulate cell function by alternative or additional mechanisms. In mast cells, for example, a variety of amphipathic cations, including mastoparan, a peptide from wasp venom, 48/80, a synthetic polycation, and polylysine, enhance secretion by a pertussis toxin-sensitive mechanism, suggesting - the involvement of a G protein. No classic cell surface receptor has been identified that could mediate the actions of these diverse agents. Furthermore, these same compounds have been shown to activate directly purified G proteins in solution or in artificial phospholipid vesicles. On the basis of these observations, it has been proposed that amphipathic cations activate G proteins and, in turn, mast cell secretion by a "receptor-independent" mechanism.
Polycations have also been shown to interact strongly with acidic phospholipids. Polylysines of varying chain lengths (20-1000 amino acids) bind to artificial phospholipid vesicles with dissociation constants in the range of 0.5 nM to 1.5 .mu.M. The binding affinity is directly related to the length of the polylysine chain, with polymers of 1000 amino acids having a K.sub.d of 0.5 nM, shorter polymers having higher Kd values, and lysine not interacting to a significant extent.
This relationship between potency and chain length is similar to that observed for the effects of polylysine 10,200, polylysine 3800, and lysine on parathyroid function.
It is possible that the binding of polycations to biomembranes produces some of their biologic actions. The permeabilization of the plasma membrane induced in some cell types by a variety of pore-forming agents, including polycations, has been postulated to be mediated by their interaction with a phosphatidylserine-like structure. In addition, the "receptor-independent" activation of purified G proteins by amphipathic cations is potentiated when these proteins are incorporated into phospholipid vesicles.
Calcium ions, in the millimolar concentration range, also produce marked changes in membrane structure. In some cases, calcium can either antagonize or potentiate the interaction of polycations with membrane lipids. These considerations raise the possibility that the actions of both polyvalent cations and polycations on parathyroid cells could involve a receptor-independent mechanism not requiring the presence of a classic, cell surface, G protein-coupled receptor. Further studies, however, are required to elucidate the molecular basis for Ca.sup.2+ sensing by this and other cell types. [Citations omitted.]
Shoback and Chen, J. Bone Mineral Res. 6 (Supplement 1) 1991, S135) and Racke et al., J. Bone Mineral Res. 6 (Supplement 1) 1991, S118) describe experiments which are said to indicate that a calcium receptor or Ca.sup.2+ sensor is present in parathyroid cells. Messenger RNA isolated from such cells can be expressed in oocytes and caused to provide those oocytes with a phenotype which might be explained by the presence of a calcium receptor protein.